G protein signaling pathways are targeted by nearly half of all pharmaceuticals, and have revolutionized the treatment of cardiovascular disease and psychiatric disorders. However, G protein signaling must be carefully regulated to ensure proper responses. To identify new regulators of the G protein signaling pathway, we have systematically evaluated 870 essential genes in yeast, using a library of repressible-promoter strains. Knock- down expression revealed several dozen new proteins required for proper signaling. Included in this group are members of the SCF (Skp-Cullin-F-Box) ubiquitin ligase complex. We determined that SCF promotes degradation of the G protein subunit (Gpa1) in vivo and catalyzes Gpa1 ubiquitination in vitro. Further, we have shown that Gpa1 is phosphorylated by Elm1, that Gpa1 phosphorylation precedes ubiquitination, and that both modifications are dynamically regulated. In this proposal we will investigate additional targets of SCF ubiquitination and of additional kinase regulators of the G protein signaling pathway. Aim 1: Identify targets of the SCF ubiquitin ligase complex. Preliminary data indicate that SCF regulates the stability of at least four pathway components, in addition to Gpa1. We will test the hypothesis that SCF ubiquitinates multiple pathway components in a coordinated manner and in this way serves as a 'master regulator' of signal output. Aim 2: Elucidate G protein regulation by AMPK kinases. Elm1 phosphorylates Gpa1, thereby promoting its ubiquitination. Elm1, Sak1 and Tos3 are protein kinases that activate AMPK (AMP-activated protein kinase) in response to glucose depletion. We will now investigate the ability of all three AMPK kinases to regulate Gpa1 during glucose depletion. We will determine when and where Gpa1 is phosphoryated, and how phosphorylation alters G protein activity, both in vivo and in vitro. Aim 3: Identify pathway targets of Mps1, and identify new pathway regulators. Mps1 is a dual specificity protein kinase that we have shown dampens pheromone activation. We will identify pathway components targeted by Mps1, and determine how phosphorylation affects their activity. Finally we will establish how additional essential genes regulate cellular signaling function. Our efforts have revealed new components of the G protein signaling pathway in yeast. Mechanisms discovered in yeast are typically recapitulated in more complex organisms, so the activities elucidated here will likely apply as well to hormone and neurotransmitter function in humans. PUBLIC HEALTH RELEVANCE: In this proposal we will define new ways that cells regulate their response to hormone signals. Detailed analysis of these processes will lay the foundation for understanding the causes and the most effective treatments of human disease.